Conventional titration involves serially diluting a liquid sample, and then analyzing the series of diluted samples to identify the most dilute sample which still evidences the tested-for parameter (the endpoint). Titration emulation is useful for performing titrations where the intensity of the initial titer is functionally related to the titration endpoint. One such application is fluorescent microscopy, such as used in fluorescent anti-nuclear antibody (FANA) testing.
For tests such as FANA, conventional titration involves serially diluting samples and then placing the diluted samples under a fluorescent microscope to manually evaluate each dilution and identify the most dilute sample which exhibits fluorescence. Unfortunately, this method is time consuming, arguous, material intensive and expensive.
Moreover, in addition to ANA concentration level, there are six discernible morphologic patterns associated with immunofluorescent staining. Some morphologic patterns are clinically significant. Accordingly, a sample must be visually examined in supplement to endpoint determination. Since titration emulation evaluates a single sample containing a screening dilution (typically 1:40) information, only one sample needs review to determine both endpoint and morphologic patterns.
As descried in the present specification, titration emulation typically produces a series of images corresponding to the initially diluted sample. Each image is formed from a combination of image component parameters, such as brightness, contrast and exposure time, which provides an effective level of intensity sensitivity. Parameters are adjusted such that each produced image emulates the effect of a successive dilution. Appropriate parameters and their settings for each titer are determined by calibrating to an acceptable standard.
The subject titration emulation in its preferred form presents a technician with a series of images analogous to the series of conventional dilutions he is accustomed to evaluating. Accordingly, a technician familiar with seeing dilutions at 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, and 1:5120 would be presented with eight images corresponding to these dilutions. The technician's job essentially remains the same: choose the image corresponding to the titer at which the tested-for material is no longer visible (the endpoint).
The above process can be automated to eliminate the technician. For example, a signal corresponding to each "dilution" may be compared by computer, with an automated cytology analysis system (such as Roche's Autocyte System described in patent application Ser. No. 08/135,640, filed Oct. 12, 1993, now abandoned the contents of which are herein incorporated by reference) being used to evaluate morphology. However, it is presently preferred to retain the use of a technician both as a quality control and to evaluate pattern morphology.
The number of dilutions may be varied by either evaluating varying numbers of standards, or interpolating and/or extrapolating results obtained from tested standards to generate "theoretical dilutions." Such theoretical dilutions can provide a continuum which may yield greater accuracy in reporting titrations.
Advantages of this new titration emulation system and method include cost reduction, higher throughput, more accurate measurement, minimal fading, and ease of use. No physical liter is required, thus fewer slides are needed. Accurate measurement is obtained because calibration reduces subjectivity and provides consistency between runs. Additionally, computer controlled exposure minimizes fading of the fluorescent material. Lastly, use of the system and method is simple, because the concepts involved are readily understood by those familiar with conventional titration analysis.